Methods of enhancing cell survival of stem cells

Abstract

The present invention relates compounds for stabilizing cells and methods of their use.

What is claimed is: 
     
       1. A method for preparing a cell having reduced differentiation in vitro, the method comprising:
 (a) obtaining cells comprising stem cells or progenitor cells; and 
 (b) contacting the cells with an amount of a compound having formula (I) sufficient to prevent or reduce the cells from differentiation as compared to cells in the absence of the compound, wherein the compound of formula (I) is as follows: 
 
       
         
           
           
               
               
           
         
       
       wherein,
 L 2  is substituted or unsubstituted C 1 -C 10  alkylene; and 
 y is an integer from 0 to 3; 
 z is an integer from 0 to 5; 
 X is —N═, —CH═ or —CR 5 ═; 
 R 1  is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; 
 R 3 , R 4  and R 5  are independently —CN, —S(O) n R 6 , —NR 7 R 8 , —C(O)R 9 , —C(O)R 11 , —NR 12 —C(O)—OR 13 , —C(O)NR 14 R 15 , —NR 16 S(O) 2 R 17 , —OR 18 , —S(O) 2 N(R 19 ) 2 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, wherein n is an integer from 0 to 2, wherein if z is greater than 1, two R 3  moieties are optionally joined together to form a substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; and 
 R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18  and R 19  are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; 
 or a racemate, diastereomer, tautomer, or a geometric isomer thereof, or a pharmaceutically acceptable salt thereof. 
 
     
     
       2. The method of  claim 1 , wherein the cells are:
 (a) in adherent cultures; 
 (b) in suspension cultures; 
 (c) single dissociated cells; or 
 (d) aggregated cells. 
 
     
     
       3. The method of  claim 1 , wherein the contacting step further comprises:
 passaging the cells in cultures comprising the compound having formula (I) for at least 5, 10, 15, or 20, or more generations. 
 
     
     
       4. The method of  claim 3 , wherein the cells:
 (a) are homogenous; 
 (b) are induced pluripotent stem cells; 
 (c) have morphology of hESCs; 
 (d) express pluripotency markers; 
 (e) maintain normal karyotype; 
 (f) are capable of self-renewal; 
 (g) are protected from cell death; 
 (h) are capable of forming teratomas; 
 (i) are capable of differentiating into three primary germ layers; 
 (j) are capable of lineage-specific differentiation; 
 (k) are capable of forming embryoid bodies after single cell dissociation; 
 (l) maintain their current state of differentiation; and/or 
 (m) have increased cell adhesion ability after single cell dissociation. 
 
     
     
       5. The method of  claim 1 , wherein the contacting step further comprises:
 (a) modulating cell surface level of E-cadherins comprising (i) stabilizing E-cadherin on cell surface; and/or (ii) inhibiting endocytosis of E-cadherins; or 
 (b) modulating integrin activity comprising (i) increasing integrin expression; and/or (ii) converting integrin into an active conformation. 
 
     
     
       6. The method of  claim 3 , wherein the cells in cultures comprising the compound having formula (I) are passaged for at least 20 generations or more generations. 
     
     
       7. The method of  claim 3 , wherein the cultures are:
 (a) chemically-defined; 
 (b) feeder-free or non-feeder cell conditioned; and/or 
 (c) animal product-free. 
 
     
     
       8. A population of isolated cells prepared according to the method of  claim 1 , wherein the isolated cells have reduced differentiation. 
     
     
       9. A composition comprising the population of isolated cells of  claim 8 . 
     
     
       10. The composition of  claim 9 , further comprising a compound of formula (I): 
       
         
           
           
               
               
           
         
       
       wherein,
 L 2  is substituted or unsubstituted C 1 -C 10  alkylene; and 
 y is an integer from 0 to 3; 
 z is an integer from 0 to 5; 
 X is —N═, —CH═ or —CR 5 ═; 
 R 1  is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; 
 R 3 , R 4  and R 5  are independently —CN, —S(O) n R 6 , —NR 7 R 8 , —C(O)R 9 , —NR 10 —C(O)R 11 , —NR 12 —C(O)—OR 13 , —C(O)NR 14 R 15 , —NR 16 S(O) 2 R 17 , —S(O) 2 N(R 19 ) 2 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, wherein n is an integer from 0 to 2, wherein if z is greater than 1, two R 3  moieties are optionally joined together to form a substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; and 
 R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18  and R 19  are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; 
 or a racemate, diastereomer, tautomer, or a geometric isomer thereof, or a pharmaceutically acceptable salt thereof. 
 
     
     
       11. A method for treating an individual by administering the composition of  claim 9  to an individual in need of treatment. 
     
     
       12. The method of  claim 11 , wherein the composition further comprises a compound of formula (I): 
       
         
           
           
               
               
           
         
       
       wherein,
 L 2  is substituted or unsubstituted C 1 -C 10  alkylene; and 
 y is an integer from 0 to 3; 
 z is an integer from 0 to 5; 
 X is —N═, —CH═ or —CR 5 ═; 
 R 1  is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; 
 R 3 , R 4  and R 5  are independently —CN, —S(O) n R 6 , —NR 7 R 8 , —C(O)R 9 , —NR 10 —C(O)R 11 , —NR 12 —C(O)—OR 13 , —C(O)NR 14 R 15 , —NR 16 S(O) 2 R 17 , —S(O) 2 N(R 19 ) 2 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, wherein n is an integer from 0 to 2, wherein if z is greater than 1, two R 3  moieties are optionally joined together to form a substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; and 
 R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18  and R 19  are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; 
 or a racemate, diastereomer, tautomer, or a geometric isomer thereof, or a pharmaceutically acceptable salt thereof. 
 
     
     
       13. The method of  claim 11 , wherein the individual has a central nervous system injury; and wherein the composition is administered in an amount sufficient to promote axonal regeneration and/or functional recovery in the injured central nervous system. 
     
     
       14. The method of  claim 11 , wherein the individual is in need of an organ, cell, or tissue transplantation; and wherein the composition is administered during and/or after transplantation in an amount sufficient to improve organ, cell, or tissue transplantation. 
     
     
       15. A method for treating an individual by administering a composition comprising a compound of formula (I) to an individual in need of treatment, wherein the compound of formula (I) is as follows: 
       
         
           
           
               
               
           
         
       
       wherein,
 L 2  is substituted or unsubstituted C 1 -C 10  alkylene; and 
 y is an integer from 0 to 3; 
 z is an integer from 0 to 5; 
 X is —N═, —CH═ or —CR 5 ═; 
 R 1  is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; 
 R 3 , R 4  and R 5  are independently —CN, —S(O) n R 6 , —NR 7 R 8 , —C(O)R 9 , —C(O)R 11 , —NR 12 —C(O)—OR 13 , —C(O)NR 14 R 15 , —NR 16 S(O) 2 R 17 , —OR 18 , —S(O) 2 N(R 19 ) 2 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, wherein n is an integer from 0 to 2, wherein if z is greater than 1, two R 3  moieties are optionally joined together to form a substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; and 
 R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18  and R 19  are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; 
 or a racemate, diastereomer, tautomer, or a geometric isomer thereof, or a pharmaceutically acceptable salt thereof. 
 
     
     
       16. The method of  claim 15 , wherein the individual has a central nervous system injury; and wherein the composition is administered in an amount sufficient to promote axonal regeneration and/or functional recovery in the injured central nervous system. 
     
     
       17. The method of  claim 15 , wherein the individual is in need of an organ, cell, or tissue transplantation; and wherein the composition is administered during and/or after transplantation in an amount sufficient to improve organ, cell, or tissue transplantation. 
     
     
       18. A method for preparing a cell having enhanced survival and/or proliferation in vitro, the method comprising:
 (a) obtaining cells comprising stem cells or progenitor cells; and 
 (b) contacting the cells with an amount of a compound having formula (I) sufficient to improve survival of the cells by at least 2-fold as compared to cells in the absence of the compound, wherein the compound of formula (I) is as follows: 
 
       
         
           
           
               
               
           
         
       
       wherein,
 L 2  is substituted or unsubstituted C 1 -C 10  alkylene; and 
 y is an integer from 0 to 3; 
 z is an integer from 0 to 5; 
 X is —N═, —CH═ or —CR 5 ═; 
 R 1  is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; 
 R 3 , R 4  and R 5  are independently —CN, —S(O) n R 6 , —NR 7 R 8 , —C(O)R 9 , —NR 10 —C(O)R 11 , —NR 12 —C(O)—OR 13 , —C(O)NR 14 R 15 , —NR 16 S(O) 2 R 17 , —OR 18 , —S(O) 2 N(R 19 ) 2 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, wherein n is an integer from 0 to 2, wherein if z is greater than 1, two R 3  moieties are optionally joined together to form a substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; and 
 R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18  and R 19  are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; 
 or a racemate, diastereomer, tautomer, or a geometric isomer thereof, or a pharmaceutically acceptable salt thereof. 
 
     
     
       19. The method of  claim 18 , wherein the cells are:
 (a) in adherent cultures; 
 (b) in suspension cultures; 
 (c) single dissociated cells; or 
 (d) aggregated cells. 
 
     
     
       20. The method of  claim 18 , wherein the contacting step further comprises:
 passaging the cells in cultures comprising the compound having formula (I) for at least 5, 10, 15, or 20, or more generations. 
 
     
     
       21. The method of  claim 20 , wherein the cells:
 (a) are homogenous; 
 (b) are induced pluripotent stem cells; 
 (c) have morphology of hESCs; 
 (d) express pluripotency markers; 
 (e) maintain normal karyotype; 
 (f) are capable of self-renewal; 
 (g) are protected from cell death; 
 (h) are capable of forming teratomas; 
 (i) are capable of differentiating into three primary germ layers; 
 (l) are capable of lineage-specific differentiation; 
 (k) are capable of forming embryoid bodies after single cell dissociation; 
 (l) maintain their current state of differentiation; and/or 
 (m) have increased cell adhesion ability after single cell dissociation. 
 
     
     
       22. The method of  claim 18 , wherein the contacting step further comprises:
 (a) modulating cell surface level of E-cadherins comprising (i) stabilizing E-cadherin on cell surface; and/or (ii) inhibiting endocytosis of E-cadherins; or 
 (b) modulating integrin activity comprising (i) increasing integrin expression; and/or (ii) converting integrin into an active conformation. 
 
     
     
       23. The method of  claim 20 , wherein the cells in cultures comprising the compound having formula (I) are passaged for at least 20 generations or more generations. 
     
     
       24. The method of  claim 20 , wherein the cultures are:
 (a) chemically-defined; 
 (b) feeder-free or non-feeder cell conditioned; and/or 
 (c) animal product-free. 
 
     
     
       25. A population of isolated cells prepared according to the method of  claim 18 , wherein the isolated cells have enhanced survival and/or proliferation. 
     
     
       26. A composition comprising the population of isolated cells of  claim 25 . 
     
     
       27. The composition of  claim 26 , further comprising a compound of formula (I): 
       
         
           
           
               
               
           
         
       
       wherein,
 L 2  is substituted or unsubstituted C 1 -C 10  alkylene; and 
 y is an integer from 0 to 3; 
 z is an integer from 0 to 5; 
 X is —N═, —CH═ or —CR 5 ═; 
 R 1  is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; 
 R 3 , R 4  and R 5  are independently —CN, —S(O) n R 6 , —NR 7 R 8 , —C(O)R 9 , —NR 10 —C(O)R 11 , —NR 12 —C(O)—OR 13 , —C(O)NR 14 R 15 , —NR 16 S(O) 2 R 17 , —S(O) 2 N(R 19 ) 2 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, wherein n is an integer from 0 to 2, wherein if z is greater than 1, two R 3  moieties are optionally joined together to form a substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; and 
 R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18  and R 19  are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; 
 or a racemate, diastereomer, tautomer, or a geometric isomer thereof, or a pharmaceutically acceptable salt thereof. 
 
     
     
       28. The method of  claim 1 , wherein the compound of formula (I) is selected from the group consisting of: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
     
     
       29. The method of  claim 1 , wherein the compound of formula (I) is selected from the group consisting of: